The penultimate step of the coagulation cascade is the Factor Xa-complex-catalyzed conversion of the zymogen prothrombin to the active enzyme thrombin. Prothrombin is a single-chain, vitamin K-dependent glycoprotein that is synthesized in the liver. It contains a gla domain, two kringle regions, an A chain, and a serine protease domain (B chain). Conversion to thrombin requires that prothrombin be cleaved in two places, removing the gla domain and kringle regions and cleaving between the A and B chains to produce the active protease, termed “α-thrombin.”
Thrombin is used therapeutically to promote hemostasis in surgery and as a component of tissue adhesives and sealants. Human and bovine thrombins, both derived from plasma, are currently approved for therapeutic use.
It would be advantageous to obtain thrombin from a recombinant source to avoid the potential for contamination that is inherent in plasma-derived products. Bovine thrombin has been associated with hemostatic abnormalities resulting from immunogenicity of the bovine thrombin itself and/or contaminant proteins (Ortel et al., Ann. Surg. 233(1):88-96, 2001; Lawson et al., Ann. Thorac. Surg. 79(3):1037-1038, 2005) and carries a “black box” warning cautioning against repeat use in patients who have developed antibodies to bovine thrombin. However, production of recombinant prothrombin has proven problematic and yields have remained low.
As an alternative to purification from plasma, thrombin can be prepared from a recombinant prethrombin (e.g., prethrombin-1) as disclosed in U.S. Pat. No. 5,476,777. Prethrombin-1 is an inactive thrombin precursor that does not contain the gla domain or the first kringle region of prothrombin, which can be produced by expression of a truncated prothrombin DNA in recombinant cells. Active thrombin is produced from prethrombin-1 by treatment with any of several activating proteases, including prothrombin activators obtained from snake venom. See, for example, Speijer et al., J. Biol. Chem. 261:13258-13267, 1986; Masci et al., Biochemistry International 17:825-835, 1988; and Morita et al., Meth. Enzym. 80:303-311, 1980.
Activation of prethrombin-1 to thrombin is complicated by the proteolytic activity of α-thrombin on prethrombin. This activity, which can reduce overall yield of α-thrombin, can be enhanced by conditions needed to stabilize the protein. There remains a need in the art for methods of efficiently activating prethrombin-1 to α-thrombin.